Browsing by Author "Sanchez, Fabiola A."

Now showing 1 - 3 of 3
  • No Thumbnail Available
    Item
    Galectin-8 induces endothelial hyperpermeability through the eNOS pathway involving S-nitrosylation-mediated adherens junction disassembly
    (2019) Zamorano, Patricia; Koning, Tania; Oyanadel, Claudia; Mardones, Gonzalo A.; Ehrenfeld, Pamela; Boric, Mauricio P.; Gonzalez, Alfonso; Soza, Andrea; Sanchez, Fabiola A.
    The permeability of endothelial cells is regulated by the stability of the adherens junctions, which is highly sensitive to kinase-mediated phosphorylation and endothelial nitric oxide synthase (eNOS)-mediated S-nitrosylation of its protein components. Solid tumors can produce a variety of factors that stimulate these signaling pathways leading to endothelial cell hyperpermeability. This generates stromal conditions that facilitate tumoral growth and dissemination. Galectin-8 (Gal-8) is overexpressed in several carcinomas and has a variety of cellular effects that can contribute to tumor pathogenicity, including angiogenesis. Here we explored whether Gal-8 has also a role in endothelial permeability. We show that recombinant Gal-8 activates eNOS, induces S-nitrosylation of p120-catenin (p120) and dissociation of adherens junction, leading to hyperpermeability of the human endothelial cell line EAhy926. This pathway involves focal-adhesion kinase (FAK) activation downstream of eNOS as a requirement for eNOS-mediated p120 S-nitrosylation. This suggests a reciprocal, yet little understood, regulation of phosphorylation and S-nitrosylation events acting upon adherens junction permeability. In addition, glutathione S-transferase (GST)-Gal-8 pull-down experiments and function-blocking beta 1-integrin antibodies point to beta 1-integrins as cell surface components involved in Gal-8-induced hyperpermeability. Endogenous Gal-8 secreted from the breast cancer cell line MCF-7 has similar hyperpermeability and signaling effects. Furthermore, the mouse cremaster model system showed that Gal-8 also activates eNOS, induces S-nitrosylation of adherens junction components and is an effective hyperpermeability agent in vivo. These results add endothelial permeability regulation by S-nitrosylation as a new function of Gal-8 that can potentially contribute to the pathogenicity of tumors overexpressing this lectin.
  • No Thumbnail Available
    Item
    S-Nitrosation of β-Catenin and p120 Catenin A Novel Regulatory Mechanism in Endothelial Hyperpermeability
    (2012) Marin, Natalie; Zamorano, Patricia; Carrasco, Rodrigo; Mujica, Patricio; Gonzalez, Francisco G.; Quezada, Claudia; Meininger, Cynthia J.; Boric, Mauricio P.; Duran, Walter N.; Sanchez, Fabiola A.
    Rationale: Endothelial adherens junction proteins constitute an important element in the control of microvascular permeability. Platelet-activating factor (PAF) increases permeability to macromolecules via translocation of endothelial nitric oxide synthase (eNOS) to cytosol and stimulation of eNOS-derived nitric oxide signaling cascade. The mechanisms by which nitric oxide signaling regulates permeability at adherens junctions are still incompletely understood.
  • No Thumbnail Available
    Item
    TNF-α-activated eNOS signaling increases leukocyte adhesion through the S- nitrosylation pathway
    (2021) Aguilar, Gaynor; Cordova, Francisco; Koning, Tania; Sarmiento, Jose; Boric, Mauricio P.; Birukov, Konstantin; Cancino, Jorge; Varas-Godoy, Manuel; Soza, Andrea; Alves, Natascha G.; Mujica, Patricio E.; Duran, Walter N.; Ehrenfeld, Pamela; Sanchez, Fabiola A.
    Nitric oxide ( NO) is a key factor in inflammation. Endothelial nitric oxide synthase (eNOS), whose activity increases after stimulation with proinflammatory cytokines, produces NO in endothelium. NO activates two pathways: 1) soluble guanylate cyclase-protein kinase G and 2) S-nitrosylation (NO-induced modification of free-thiol cysteines in proteins). S-nitrosylation affects phosphorylation, localization, and protein interactions. NO is classically described as a negative regulator of leukocyte adhesion to endothelial cells. However, agonists activating NO production induce a fast leukocyte adhesion, which suggests that NO might positively regulate leukocyte adhesion. We tested the hypothesis that eNOS-induced NO promotes leukocyte adhesion through the S-nitrosylation pathway. We stimulated leukocyte adhesion to endothelium in vitro and in vivo using tumor necrosis factor-alpha (TNF-alpha) as proinflammatory agonist. ICAM-1 changes were evaluated by immunofluorescence, subcellular fractionation, immunoprecipitation, and fluorescence recovery after photobleaching (FRAP). Protein kinase C sigma (PKC sigma) activity and S-nitrosylation were evaluated by Western blot analysis and biotin switch method, respectively. TNF-alpha, at short times of stimulation, activated the eNOS S-nitrosylation pathway and caused leukocyte adhesion to endothelial cells in vivo and in vitro. TNF-alpha-induced NO led to changes in ICAM-1 at the cell surface, which are characteristic of clustering. TNF-alpha-induced NO also produced S-nitrosylation and phosphorylation of PKCf, association of PKCf with ICAM-1, and ICAM-1 phosphorylation. The inhibition of PKCf blocked leukocyte adhesion induced by TNF-alpha. Mass spectrometry analysis of purified PKCf identified cysteine 503 as the only S-nitrosylated residue in the kinase domain of the protein. Our results reveal a new eNOS S-nitrosylation-dependent mechanism that induces leukocyte adhesion and suggests that S-nitrosylation of PKCf may be an important regulatory step in early leukocyte adhesion in inflammation.